1. Field of Invention
The current invention relates to confocal spectroscopy systems and methods, and more particularly to cylindrical illumination confocal spectroscopy systems and methods.
2. Discussion of Related Art
Single molecule detection (SMD) allows the study of molecular properties without the bias of ensemble averaging. Although methods using scanning probe, resonant, and electrical sensors are being developed (Craighead, H. 2006. Future lab-on-a-chip technologies for interrogating individual molecules. Nature 442:387-393), it can also be performed using confocal spectroscopy, an optical detection method in which a collimated laser beam is focused into a diffraction-limited spot about 1 femtoliter in volume and used to excite single fluorescent molecules. While biomolecules are often tethered to solid substrates for in depth study of molecular dynamics, continuous flow systems can offer higher throughput and other advantages for quantitative applications. Herein, SMD will refer to confocal spectroscopy of molecules in free solution under continuous flow. SMD can be ideally suited as a platform for the detection of rare biomolecules such as nucleic acids (Li, H., L. Ying, J. J. Green, S. Balasubramanian, and D. Klenerman. 2003. Ultrasensitive coincidence fluorescence detection of single DNA molecules. Anal. Chem. 75:1664-1670; Camacho, A., K. Korn, M. Damond, J. F. Cajot, E. Litborn, B. Liao, P. Thyberg, H. Winter, A. Honegger, P. Gardellin, and R. Rigler. 2004. Direct quantification of mRNA expression levels using single molecule detection. J. Biotechnol. 107:107-114; Wabuyele, M. B., H. Farquar, W. Stryjewski, R. P. Hammer, S. A. Soper, Y. W. Cheng, and F. Barany. 2003. Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes. J. Am. Chem. Soc. 125:6937-6945; Zhang, C. Y., S. Y. Chao, and T. H. Wang. 2005. Comparative quantification of nucleic acids using single-molecule detection and molecular beacons. The Analyst 130:483-488), proteins, and small ligands (Pons, T., I. L. Medintz, X. Wang, D. S. English, and H. Mattoussi. 2006. Solution-phase single quantum dot fluorescence resonance energy transfer. J. Am. Chem. Soc. 128:15324-15331), the characterization of biomolecular interactions and molecular processes (Lipman, E. A., B. Schuler, O. Bakajin, and W. A. Eaton. 2003. Single-molecule measurement of protein folding kinetics. Science 301:1233-1235; Ha, T., I. Rasnik, W. Cheng, H. P. Babcock, G. H. Gauss, T. M. Lohman, and S. Chu. 2002. Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419:638-641), DNA sizing (Habbersett, R. C., and J. H. Jett. 2004. An analytical system based on a compact flow cytometer for DNA fragment sizing and single-molecule detection. Cytometry A 60:125-134), and pathogen detection (Agrawal, A., R. A. Tripp, L. J. Anderson, and S. Nie. 2005. Real-time detection of virus particles and viral protein expression with two-color nanoparticle probes. J. Virol. 79:8625-8628).
Although in principle SMD can be highly quantitative, its current implementations limit its accuracy, throughput, and practical applicability. The minute size of the SMD observation volume enables high signal-to-noise ratio detection of even single fluorescent molecules due to highly suppressed background levels. However, the diffraction-limited observation volume that enables SMD also significantly hampers its application in quantification and burst parameter determination. Since the observation volume in standard SMD is typically much smaller than the channel used for molecular transport, a condition of low mass detection efficiency is created where the large majority of molecules escape detection. We define the mass detection efficiency as the total proportion of molecules flowing through the channel that are detected. These mass detection efficiencies are usually 1% or less (Haab, B. B., and R. A. Mathies. 1999. Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams. Anal. Chem. 71:5137-5145). For example, assuming 1) that all molecules passing within the observation volume are detected, 2) a radially symmetric, ellipsoidal, confocal observation volume with 1/e2 radii of 0.5×1 μm, and 3) detection within a 100 μm ID microcapillary, the resultant mass detection efficiency would be less than 0.05%. This necessitates extended data acquisition times and increased sample volumes for the detection of rare molecules (Wang, T. H., Y. H. Peng, C. Y. Zhang, P. K. Wong, and C. M. Ho. 2005. Single-molecule tracing on a fluidic microchip for quantitative detection of low-abundance nucleic acids. J. Am. Chem. Soc. 127:5354-5359). In addition, since the observation volume profile is Gaussian in shape and highly non-uniform, a molecule's specific trajectory through the detection region will have a large influence on the emitted and collected fluorescence bursts, adding significant variability and uncertainty to not only the burst parameters but also their rate of detection.
The majority of approaches to rectify these short-comings have centered around controlling the molecular trajectory using either hydrodynamic (de Mello, A. J., and J. B. Edel. 2007. Hydrodynamic focusing in microstructures: Improved detection efficiencies in subfemtoliter probe volumes. J. Appl. Phys. 101:084903; Werner, J. H., E. R. McCarney, R. A. Keller, K. W. Plaxco, and P. M. Goodwin. 2007. Increasing the resolution of single pair fluorescence resonance energy transfer measurements in solution via molecular cytometry. Anal. Chem. 79:3509-3513) or electrokinetic (Haab, B. B., and R. A. Mathies. 1999. Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams. Anal. Chem. 71:5137-5145; Wang, T. H., Y. H. Peng, C. Y. Zhang, P. K. Wong, and C. M. Ho. 2005. Single-molecule tracing on a fluidic microchip for quantitative detection of low-abundance nucleic acids. J. Am. Chem. Soc. 127:5354-5359; Schrum, D. P., C. T. Culbertson, S. C. Jacobson, and J. M. Ramsey. 1999. Microchip flow cytometry using electrokinetic focusing. Anal. Chem. 71:4173-4177) forces as well as nanochannel confinement (Foquet, M., J. Korlach, W. Zipfel, W. W. Webb, and H. G. Craighead. 2002. DNA fragment sizing by single molecule detection in submicrometer-sized closed fluidic channels. Anal. Chem. 74:1415-1422; Dorre, K., J. Stephan, M. Lapczyna, M. Stuke, H. Dunkel, and M. Eigen. 2001. Highly efficient single molecule detection in microstructures. J. Biotechnol. 86:225-236; Lyon, W. A., and S. Nie. 1997. Confinement and Detection of Single Molecules in Submicrometer Channels. Anal. Chem. 69:3400-3405). However, these approaches have limitations in their practical application due to effectiveness, throughput limitations, and ease of use, for example. Therefore, there remains a need for improved single molecule detection systems and methods.